Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

Muscle,Skin and Core Temperature after ?110°C Cold Air and 8°C Water Treatment

The aim of this investigation was to elucidate the reductions in muscle, skin and core temperature following exposure to −110°C whole body cryotherapy (WBC), and compare these to 8°C cold water immersion (CWI). Twenty active male subjects were randomly assigned to a 4-min exposure of WBC or CWI. A minimum of 7 days later subjects were exposed to the other treatment. Muscle temperature in the right vastus lateralis (n = 10); thigh skin (average, maximum and minimum) and rectal temperature (n = 10) were recorded before and 60 min after treatment. The greatest reduction (P<0.05) in muscle (mean ± SD; 1 cm: WBC, 1.6±1.2°C; CWI, 2.0±1.0°C; 2 cm: WBC, 1.2±0.7°C; CWI, 1.7±0.9°C; 3 cm: WBC, 1.6±0.6°C; CWI, 1.7±0.5°C) and rectal temperature (WBC, 0.3±0.2°C; CWI, 0.4±0.2°C) were observed 60 min after treatment. The largest reductions in average (WBC, 12.1±1.0°C; CWI, 8.4±0.7°C), minimum (WBC, 13.2±1.4°C; CWI, 8.7±0.7°C) and maximum (WBC, 8.8±2.0°C; CWI, 7.2±1.9°C) skin temperature occurred immediately after both CWI and WBC (P<0.05). Skin temperature was significantly lower (P<0.05) immediately after WBC compared to CWI. The present study demonstrates that a single WBC exposure decreases muscle and core temperature to a similar level of those experienced after CWI. Although both treatments significantly reduced skin temperature, WBC elicited a greater decrease compared to CWI. These data may provide information to clinicians and researchers attempting to optimise WBC and CWI protocols in a clinical or sporting setting.     

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.     

Physical and Perceptual Cooling with Beverages to Increase Cycle Performance in a Tropical Climate

Purpose

This study compares the effects of neutral temperature, cold and ice-slush beverages, with and without 0.5% menthol on cycling performance, core temperature (T_co) and stress responses in a tropical climate (hot and humid conditions).

Methods

Twelve trained male cyclists/triathletes completed six 20-km exercise trials against the clock in 30.7°C±0.8°C and 78%±0.03% relative humidity. Before and after warm-up, and before exercise and every 5 km during exercise, athletes drank 190 mL of either aromatized (i.e., with 0.5 mL of menthol (5 gr/L)) or a non-aromatized beverage (neutral temperature: 23°C±0.1°C, cold: 3°C±0.1°C, or ice-slush: −1°C±0.7°C). During the trials, heart rate (HR) was continuously monitored, whereas core temperature (T_co), thermal comfort (TC), thermal sensation (TS) and rate of perceived exertion (RPE) were measured before and after warm-up, every 5 km of exercise, and at the end of exercise and after recovery.

Results

Both the beverage aroma (P<0.02) and beverage temperature (P<0.02) had significant and positive effects on performance, which was considerably better with ice-slush than with a neutral temperature beverage, whatever the aroma (P<0.002), and with menthol vs non-menthol (P<0.02). The best performances were obtained with ice-slush/menthol and cold/menthol, as opposed to neutral/menthol. No differences were noted in HR and T_co between trials.

Conclusion

Cold water or ice-slush with menthol aroma seems to be the most effective beverage for endurance exercise in a tropical climate. Further studies are needed to explore its effects in field competition.     

Raw Cow Milk Bacterial Population Shifts Attributable to Refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (~150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Deposition of Aliphatic Suberin Monomers and Associated Alkanes during Aging of Solanum tuberosum L. Tuber Tissue at Different Temperatures

The effect of temperature on suberization of potato tuber tissue was measured by diffusive resistance and quantitative chemical procedures. The optimum temperature for formation of aliphatic suberin monomers and development of resistance to water vapor conduction was 26.4°C whereas alkane synthesis was optimal at 18.6°C. Low temperatures (<16.6°C) reduced suberin monomer production more than alkane synthesis.     

Hyperthermia in Human Ischemic and Hemorrhagic Stroke: Similar Outcome,Different Mechanisms

Hyperthermia is a predictor of poor outcome in ischemic (IS) and intracerebral hemorrhagic (ICH) stroke. Our aim was to study the plausible mechanisms involved in the poor outcome associated to hyperthermia in stroke. We conducted a case-control study including patients with IS (n = 100) and ICH (n = 100) within the first 12 hours from symptom onset. Specifically, IS and ICH patients were consecutively included into 2 subgroups, according to the highest body temperature within the first 24 hours: Tmax <37.5°C and Tmax ≥37.5°C, up to reach 50 patients per subgroup of temperature for both IS and ICH patients. Body temperature was determined at admission and every 4 hours during the first 48 hours. Main outcome variable was poor functional outcome (modified Rankin scale score >2) at 3 months. Serum levels of glutamate and active MMP-9 were measured at admission. Our results showed that Tmax ≥37.5°C within the first 24 hours was independently associated with poor outcome in both IS (OR, 12.43; 95% CI, 3.73–41.48; p<0.0001) and ICH (OR, 4.29; 95% CI, 1.32–13.91; p = 0.015) after adjusting for variables with a proven biological relevance for outcome. However, when molecular markers levels were included in the logistic regression model, we observed that glutamate (OR, 1.01; 95% CI, 1.00–1.02; p = 0.001) and infarct volume (OR, 1.06; 95% CI, 1.01–1.10; p = 0.015) were the only variables independently associated to poor outcome in IS, and active MMP-9 (OR, 1.04; 95% CI, 1.00–1.08; p = 0.002) and National Institute of Health Stroke Scale (NIHSS) at admission (OR, 1.29; 95% CI, 1.13–1.49; p<0.0001) in ICH. In conclusion, these results suggest that although the outcome associated to hyperthermia is similar in human IS and ICH, the underlying mechanisms may be different.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth

Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

A general approach to antibody thermostabilization

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

A Transport Model for Estimating the Time Course of ERK Activation in the C.?elegans Germline

The Caenorhabditis elegans germline is a well-studied model system for investigating the control of cell fate by signaling pathways. Cell signals at the distal tip of the germline promote cell proliferation; just before the loop, signals couple cell maturation to organism-level nutrient status; at the proximal end of the germline, signals coordinate oocyte maturation and fertilization in the presence of sperm. The latter two events require dual phosphorylation and activation of ERK, the effector molecule of the Ras/MAPK cascade. In C. elegans, ERK is known as MPK-1. At this point, none of today’s methods for real-time monitoring of dually phosphorylated MPK-1 are working in the germline. Consequently, quantitative understanding of the MPK-1-dependent processes during germline development is limited. Here, we make a step toward advancing this understanding using a model-based framework that reconstructs the time course of MPK-1 activation from a snapshot of a fixed germline. Our approach builds on a number of recent studies for estimating temporal dynamics from fixed organisms, but takes advantage of the anatomy of the germline to simplify the analysis. Our model predicts that the MPK-1 signal turns on ∼30 h into germ cell progression and peaks ∼7 h later.     

Persistence of F-Specific RNA Coliphages in Surface Waters from a Produce Production Region along the Central Coast of California

F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence–all prototype and environmental strains survived significantly longer at 10°C compared to 25°C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli F_amp, all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25°C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25°C was QB>MS2>SP>GA and at 10°C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25°C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

The core interthreshold zone during exposure to red and blue light

Background

This study tested the hypothesis that the core interthreshold zone (CIZ) changes during exposure to red or blue light via the non-visual pathway, because it is known that light intensity affects the central nervous system. We conducted a series of human experiments with 5 or 10 male subjects in each experiment.

Methods

The air temperature in the climatic chamber was maintained at 20 to 24°C. The subjects wore suits perfused with 25°C water at a rate of 600 cm³/min. They exercised on an ergometer at 50% of their maximum work rate for 10 to 15 minutes until sweating commenced, and then remained continuously seated without exercise until their oxygen uptake increased. The rectal temperature and skin temperatures at four sites were monitored using thermistors. The sweating rate was measured at the forehead with a sweat rate monitor. Oxygen uptake was monitored with a gas analyzer. The subjects were exposed to red or blue light at 500 lx and 1000 lx in both summer and winter.

Results

The mean CIZs at 500 lx were 0.23 ± 0.16°C under red light and 0.20 ± 0.10°C under blue light in the summer, and 0.19 ± 0.20°C under red light and 0.26 ± 0.24°C under blue light in the winter. The CIZs at 1000 lx were 0.18 ± 0.14°C under red light and 0.15 ± 0.20°C under blue light in the summer, and 0.52 ± 0.18°C under red light and 0.71 ± 0.28°C under blue light in the winter. A significant difference (P <0.05) was observed in the CIZs between red and blue light at 1000 lx in the winter, and significant seasonal differences under red light (P <0.05) and blue light (P <0.01) were also observed at 1000 lx.

Conclusions

The present study demonstrated that dynamic changes in the physiological effects of colors of light on autonomic functions via the non-visual pathway may be associated with the temperature regulation system.